Hepatitis C virus (HCV) infections are a significant threat to human health. Currently, no vaccine exists against infection and/or the liver disease caused by HCV. Because a cell-culture system for the propagation of the virus is not available, modified live-virus or inactivated vaccine development has been impossible. The objective of the proposal presented here is to demonstrate that the equine herpesvirus type 1 (EHV-1) vaccine strain RacH can be developed into a vector that is able to efficiently deliver sequences encoding HCV structural proteins. We will also test, which route of application of the EHV-1 vector results in a significant humoral and cellular immune response against HCV. EHV-1 is a novel vaccine vector given its efficient entry into primary human peripheral blood mononuclear cells (PBMC), including professional antigen-presenting cells, its ability to infect at low particle per cell numbers, and the possibility to engineer extremely safe replication-deficient EHV-1 that can accommodate large pieces of foreign DNA. In addition, pre-existing immunity against EHV-1 in humans is absent. Specific aim 1. To construct and characterize EHV-1 recombinants expressing HCV structural proteins C/E1 to p7. Recombinant viruses will be generated using the infectious bacterial artificial chromosome (BAG) clone of RacH (pRacH). We shall generate EHV-1 expressing the capsid to p7 (C-p7) or the El to p7 (El-p7) region of HCV. The HCV constructs will be inserted in two different genomic loci of the RacH genome, namely the unique-long or unique-short region, to exclude positional effects of foreign gene insertion. The same HCV structural gene region will also be inserted into an extremely safe replication-deficient RacH virus, which has a deletion in the sole and essential immediate-early gene and is unable to replicate or produce any EHV-1 proteins unless propagated in a complementing cell line. Specific aim 2. To evaluate the immunogenic properties of recombinant EHV-1 expressing HCV proteins. The pRacH-based viruses expressing the HCV structural proteins will be tested for the induction of ELISA and neutralizing antibodies as well as cell-mediated cytotoxic (CTL) responses in mice. The tests will include ELISPOT and CTL assays in the context of a human class I allele using HLA-A2.1 transgenic mice.